Sonosynthetic Cyanobacteria Oxygenation for Self‐Enhanced Tumor‐Specific Treatment

Abstract Photosynthesis, essential for life on earth, sustains diverse processes by providing nutrition in plants and microorganisms. Especially, photosynthesis is increasingly applied in disease treatments, but its efficacy is substantially limited by the well‐known low penetration depth of external light. Here, ultrasound‐mediated photosynthesis is reported for enhanced sonodynamic tumor therapy using organic sonoafterglow (ultrasound‐induced afterglow) nanoparticles combined with cyanobacteria, demonstrating the proof‐of‐concept sonosynthesis (sonoafterglow‐induced photosynthesis) in cancer therapy. Chlorin e6, a typical small‐molecule chlorine, is formulated into nanoparticles to stimulate cyanobacteria for sonosynthesis, which serves three roles, i.e., overcoming the tissue‐penetration limitations of external light sources, reducing hypoxia, and acting as a sonosensitizer for in vivo tumor suppression. Furthermore, sonosynthetic oxygenation suppresses the expression of hypoxia‐inducible factor 1α, leading to reduced stability of downstream SLC7A11 mRNA, which results in glutathione depletion and inactivation of glutathione peroxidase 4, thereby inducing ferroptosis of cancer cells. This study not only broadens the scope of microbial nanomedicine but also offers a distinct direction for sonosynthesis.


Synthesis of NPs-Ce6
A bottom-up approach (nanoprecipitation) is used to synthesize the NPs-Ce6 from the homogeneously dissolved tetrahydrofuran (THF) solution of Ce6.Briefly, PEG-b-PPG-b-PEG (20 mg mL -1 ) and Ce6 (0.15 mg mL -1 ) were dissolved into THF solution (5 mL).The mixture was then injected into ultrapure water (45 mL) under magnetic stirring for 30 min.Then THF was evaporated with a rotary evaporator.Finally, the resulting solution of NPs-Ce6 was received by ultracentrifugation with 30 K centrifugal filter units (Millipore) and the concentrated solution of NPs-Ce6 was diluted with 1 × PBS buffer.The obtained nanoparticle dispersion was stored in the dark at 4 °C.

Apparatus and characterization
Transmission electron microscopy (TEM) was tested by JEM-F200 (JEOL, Japan).Scanning electron microscopy (SEM) was tested by JSM-6700F (JEOL, Japan).DLS and Zeta potential were tested by nanosonosensitizer size analyzer (Zetasizer 3000HS, Malvern Instruments, UN).Ultraviolet-visible (UV-vis) absorbance spectra were performed on a UV-1800 UV-vis scanning spectrometer.Fluorescence spectra measurement was carried out on a F-7000 spectrofluorimeter.ROS were identified by an EMX plus-6/1 ESR spectrometer.The confocal laser scanning microscopy (CLSM) images were obtained by a CLSM 710 microscope.The sonoafterglow images were captured on a VISQUE In-Vivo Smart-LF imaging system.Flow cytometry was performed by using a Becton-Dickinson spectrophotometer.The cell viability was measured by SpectraMax iD5 microplate reader.

Cell culture
4T1 murine breast cancer cell lines and 3T3 murine embryonic fibroblast cell lines were purchased from the cell bank of Chinese Academy of Sciences (CBCAS, Shanghai).4T1 cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ in a humidified atmosphere with 5% CO2.

Cellular uptake
4T1 cells were seeded into confocal bottom-dishes (cell density = 10 5 cells per disk) and cultured.Then the cell culture medium was replaced with fresh culture medium containing NPs-Ce6 (400 µg mL -1 ) for 0, 2, 4 and 8 h, respectively.The treated 4T1 cells were washed twice with PBS under CLSM observation.

Cell apoptosis analysis
The 4T1 cells were seeded at a density of 1×10 5 cells/well into 6-well plates and cultured with RPMI-1640 medium containing 10% FBS for 24 h.The cells were co-incubated with different treatment including for 12 h.For treatment groups, the cells were exposed to the US for sonication for 5 min (1 MHz, 1.2 W cm -2 , 50% duty cycle), and cultured for another 12 h.Then trypsinised, washed with medium and centrifuged at 2000 rpm for 5 min.The cells were resuspended in 1 mL binding buffer containing Annexin V-FITC (10 μM) and PI (10 μM).The cells were incubated for 15 min in a dark environment.Lastly, the results were analyzed by flow cytometry.

In vitro iron assessment
The cytosolic iron level was measured by calcein acetoxymethyl ester (CAL-AM).The membrane-permeant CAL-AM could be loaded into cells swiftly and cleaved to the fluorescent substance CAL, which can be quenched by permeant iron (+3 or +2) chelators.4T1 cells were incubated fresh culture medium containing NPs-Ce6 and NPs-Ce6 + cyanobacteria (NPs-Ce6: 400 µg mL -1 , cyanobacteria: 5 × 10 7 cell mL -1 ) in 24-well plates for 12 h.After the cells were irradiated with the sonication for 5 min (1 MHz, 1.2 W cm -2 , 50% duty cycle).Then, the treated cells were supplemented with CAL-AM (0.2 μM) for 15 min in serumfree medium.Afterward, the cells were harvested and resuspended in PBS (400 μL) for flow cytometry analysis.The mean fluorescence intensity of the different groups was inversely proportional to the labile iron content.

In vitro cellular oxygenation detection
The cellular oxygenation in vitro was achieved by the confocal observations using the hypoxia-sensitive fluorescence probe Ru(dpp)3Cl2.4T1 cells were seeded onto the confocal disk at a cell density of 10 5 cells/disk.After allowing the cells to attach for 12 h, they were placed in a hermetically sealed culture bag with a commercial oxygen-depriving catalyst for another 12 h.Cells were then incubated with Ru(dpp)3Cl2 (10 μg mL -1 in full 1640 medium) in a hermetic environment for 3 h.After gently rinsing with cold PBS, the previous medium was replaced with 1 mL of full 1640 medium containing NPs-Ce6 and NPs-Ce6 + cyanobacteria (NPs-Ce6: 400 µg mL -1 , cyanobacteria: 5 × 10 7 cell mL -1 ).4T1 cells were exposed to US irradiation for 5 min (1 MHz, 1.2 W cm -2 , 50% duty cycle).After co-incubating of 2 h in the dark, the cells were observed under a confocal microscope (Ex: 488 nm, Em: 610 nm).

Western blot analysis
4T1 cells were seeded into 6-well plates and cultured for 24 h.Then, 4T1 cells were cultured with fresh culture medium containing NPs-Ce6 (400 μg mL -1 ) and NPs-Ce6 (400 μg mL -1 ) + PCC (5 × 10 7 cell mL -1 ) for 4 h.Next, 4T1 cells were received with or without US irradiation for 5 min (1 MHz, 1.2 W cm -2 , 50% duty cycle), and cultured for another 6 h.Then 4T1 cells were washed with PBS and lysed to collect the protein.The PVDF membranes were blocked with 5% nonfat dry milk at room temperature for 1 h in the decoloring shaker and then incubated with anti-HIF-1α, anti-SLC7A11, anti-ferritin and anti-GPX4 overnight at 4 °C.The membranes were washed for three times in the decoloring shaker.Subsequently, the membranes were incubated with secondary antibodies for 30 min and washed for three times in the decoloring shaker subsequently.Enhanced chemiluminescence (ECL) reagent was then added and reacted for 1-2 min.The membranes were exposed in a darkroom and took images.

Animal models
All animal experiments were performed in strict compliance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai University (Approval number: ECSHU 2022-053).Female BALB/c mice and female nude mice (6-8 weeks old) were purchased from Shanghai Legen Biotechnology Co., Ltd., and raised in Specific pathogen free class environment facility.The laboratory mice were housed under controlled conditions, namely 20-23 ℃ (± 0.5 ℃), 50-70% (± 10%) relative humidity, and a 12 h:12 h light:dark cycle.Water and food were available ad libitum for all animals.

Statistical analysis
All the data in the experiments were presented as mean ± SD and were repeated at least three times.Analysis of variance (ANOVA) was used for multiple comparisons, and two-tail Student's t-test was used for two-group comparisons.All statistical analyses were carried out using GraphPad Prism 8.0.2.The threshold for statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure
Figure S1.a) Sonoafterglow intensity of NPs-Ce6 after US irradiation for 2 min, and re-irradiation for 2 min.b) Time-dependent sonoafterglow intensity of NPs-Ce6 (n = 3).Data are presented as mean values ± SD.

Figure S3 .
Figure S3.Schematic illustration showing the induction and detection of afterglow from NPs-Ce6 through chicken breast tissues following US or laser irradiation (Created with BioRender.com).

Figure S6 .
Figure S6.Standard curve of PCC at different concentrations.

Figure S8 .
Figure S8.The growth curve of PCC in BG11 medium and 1640 medium or after treatment with different concentrations NPs-Ce6 in PBS (pH 6.4).

Figure S12 .
Figure S12.Relative viabilities of 4T1 cancer cells after treatment with NPs-Ce6 at different concentrations for 24 and 48 h (n = 3).Data are presented as mean values ± SD.

Figure S21 .
Figure S21.Relative viabilities of 4T1 cancer cells with a) Fer-1 and b) DFO at different concentrations (n = 4).Data are presented as mean values ± SD.